1. BET1-Like Polypeptides
Gregorio Hueros et. al. (Plant Cell, Vol. 7, 747-757, 6/1995 Am. Soc. Plant Physiol.) disclosed a cDNA clone, BET1 (for basal endosperm transfer layer), isolated from a cDNA bank prepared from 10-days after pollination (DAP) maize endosperm mRNA. BET1 mRNA showed to encode a 7-kD cell wall polypeptide. Both the mRNA and protein were restricted in their distribution to the basal endosperm transfer layer and were not expressed elsewhere in the plant. BET1 expression commenced at 9 DAP, reached a maximum between 12 and 16 DAP, and declined after 16 DAP. The initial accumulation of the BET1 polypeptide reached a plateau by 16 DAP and declined thereafter, becoming undetectable by 20 DAP. The antibody raised against the BET1 protein reacted with a number of polypeptides of higher molecular mass than the BET1 monomer. Most of these were present in cytosolic fractions and were found in nonbasal cell endosperm extracts, but three species appeared to be basal cell specific. This result and the reactivity of exhaustively extracted cell wall material with the BET1 antibody suggest that a fraction of the protein is deposited in a covalently bound form in the extracellular matrix. It was proposed that BET1 protein plays a role in the structural specialization of the transfer cells. In addition, BET1 provides a new molecular marker for the development of this endosperm domain.
2. Calreticulin Polypeptides
Calcium plays an essential role in multiple signal transduction pathways both in plants and in animals. Cytoplasmic calcium concentrations are tightly regulated at 100-200 nM but higher levels, in the range of micro- and milli-molar are found in subcellular organelles. In plants calcium is an also a micronutrient.
Calreticulin (CRT), a protein involved in the modulation of the ER (endoplasmic reticulum) Ca2+(Calcium)-ATPase, is found in all eukaryotes. Studies in mammalians filed have elucidated the structure of the CRT proteins and a number of key physiological functions, including control of cell adhesion and signal transduction through calcium-binding and quality control of protein folding and posttranscriptional modifications (Michalak. Biochem J. 2009 417(3):651-66).
Structurally CRT proteins are characterized by three distinct domains: a globular neutral N-domain, a proline-rich P-domain, and a polyacidic C-domain. CRT also has an N-terminal signal peptide sequence and an ER retention motif in the C-domain. The P-domain is responsible for the high-affinity (in the order of Kd 1.6 micromolar) and low-capacity Ca2+ binding while the C-domain is responsible for the low-affinity (in the order of Kd 0.3-2 mM) and high-capacity Ca2+ binding. CRT polypeptides include an N-terminal signal sequence and an ER-retention motif in the C-domain. Within the P-domain, there are two types of triplicate repeated motifs that are highly conserved among various animal species. However, the C-domain is less conserved than other domains of CRT. Four amino acid residues at the tip of the “extended arm” of the P-domain are critical in the chaperone function of CRT. The C-domain is involved n the Ca2+ storage in the lumen of the ER (Michalak. Biochem J. 1992, 285 (Pt 3):681-92.).
In plants, CRT proteins share same structural features and similar Ca2+ binding proteins as their animal counterparts. Phylogenetic studies revealed that plant CRT fall into two evolutionary related groups, the so called CRT1/2 and CRT3. CRT1/2 are often localized to the plasmodesmata of the cell. Plant CRT have been proposed to play a role in regeneration, gravitropism, signal transduction, and regulation of stress tolerance (Christensen et al. 2008, Plant Cell Physiol. 49(6): 912-924).
BrCRT1, a CRT form Brassica rapa when expressed in transgenic tobacco plants displayed no obvious phenotypic differences in appearance, time of flowering, or seed production when grown to maturity in soil and a weak growth inhibition of seedlings (Jin at al. 2005 Transgenic Res. 14(5):619-26).
3. tRNA Dihydrouridine Synthase 1-Like Polypeptides (DUS1L Polypeptides)
In translation, transfer RNA is the central adapter molecule as it physically links the genetic information of messenger RNA, and the addition of correctly ordered amino acids to a growing polypeptide chain. One of the structural features of tRNA is the presence of a wide variety of post-transcriptionally modified RNA bases. Dihydrouridine is one of the most abundant modified tRNA bases in prokaryotes and eukaryotes. It differs from uridine only by the reduction of uridine's carbon-carbon double bond (non-aromatic base), and is found almost exclusively at preferred positions in the D-loop of tRNA, which can further contain varying numbers of dihydrouridine residues (Bishop et al. (2002) 277(28): 25090-25095). The most likely chemical role of dihydrouridine is to enhance the conformational flexibility of tRNA, and thus improve the translational efficiencies.
The family of dihydrouridine synthase (DUS) enzymes, which catalyze the modification of uridine to dihydrouridine, has been identified in Saccharomyces cerevisiae and E. coli (Bishop et al, supra). DUSs comprise a discrete gene family (3 members in E. coli YjbN, YhdG, and Yohl, at least 4 members in yeast YML080w or DUS1, YNR015w, YLR405w, and YLR401c), allowing putative DUS genes from other organisms to be proposed based on sequence homology. Such homologs have been found for example in human, chimpanzee, dog, cow, mouse, hicken, zebrafish, fruit fly, mosquito, C. elegans, rice, and P. falciparum. In the Arabidopsis genome, at least 3 genes have been identified as potentially encoding DUS enzymes (AT3G49640, AT4G38890, AT5G67220 or DUS1 like). One of these genes encodes a polypepyide with higher similarity to the DUS1 enzyme, and is therefore called DUS1 like (DUS1L) enzyme.
In international application WO 02/66660 “Method for identifying herbicidally active substances” a nucleic acid sequence is described encoding a DUS1L polypeptide (SEQ ID NO: 84), and constructs comprising this sequence. Transgenic plants lacking the gene product present significantly delayed growth and/or completely stunted growth at the embryonic stage of Arabidopsis thaliana. The invention relates to the use of said genes and the gene products coded thereby for discovering novel herbicides.
Surprisingly, it has now been found that increasing expression in a plant of a nucleic acid sequence encoding a DUS1L polypeptide as defined herein, gives plants having increased yield-related traits relative to control plants.
According to one embodiment, there is provided a method for increasing yield-related traits in plants relative to control plants, comprising increasing expression in a plant of a nucleic acid sequence encoding a DUS1L polypeptide as defined herein. The increased yield-related traits comprise one or more of: increased aboveground biomass, increased seed yield per plant, increased number of filled seeds, and increased total number of seeds.
4. ES43-Like Polypeptides
The BAH (bromo-adjacent homology) family contains proteins such as eukaryotic DNA (cytosine-5) methyltransferases, the origin recognition complex 1 (Orc1) proteins, as well as several proteins involved in transcriptional regulation. The BAH domain appears to act as a protein-protein interaction module specialised in gene silencing, as suggested for example by its interaction within yeast Orc1p with the silent information regulator Sir1p. The BAH module might therefore play an important role by linking DNA methylation, replication and transcriptional regulation (FEBS Lett. 1999 March 5; 446(1):189-93).
PHD domains are protein Zinc finger domains that fold into an interleaved type of Zn-finger chelating 2 Zn ions in a similar manner to that of the RING and FYVE domains (Pascual et al. J Mol Biol 2000; 304:723-729). Zinc finger (Znf) domains are relatively small protein motifs that bind one or more zinc atoms, and which usually contain multiple finger-like protrusions that make tandem contacts with their target molecule. Their binding properties depend on the amino acid sequence of the finger domains and of the linker between fingers, as well as on the higher-order structures and the number of fingers. Znf domains are often found in clusters, where fingers can have different binding specificities. There are many superfamilies of Znf motifs, varying in both sequence and structure. They display considerable versatility in binding modes, even between members of the same class (e.g. some bind DNA, others protein), suggesting that Znf motifs are stable scaffolds that have evolved specialised functions. For example, Znf-containing proteins function in gene transcription, translation, mRNA trafficking, cytoskeleton organisation, epithelial development, cell adhesion, protein folding, chromatin remodelling and zinc sensing. Zinc-binding motifs are stable structures, and they rarely undergo conformational changes upon binding their target.
The PHD (homeodomain) zinc finger domain which is a C4HC3 zinc-finger-like motif found in nuclear proteins is thought to be involved in chromatin-mediated transcriptional regulation. The PHD finger motif is reminiscent of, but distinct from the C3HC4 type RING finger (Aasland et al. Trends Biochem Sci. 1995 February; 20(2):56-9).
A number of plant proteins comprising both BAH and PHD finger domains have been described. For Example the ES43 protein of Balery (Speulman and Salamini Plant Sci.
106, 91-98 (1995), SHL (Mussig et al. Mol Gen Genet. 2000 November; 264(4):363-70) and EBS (Pi{umlaut over (n)}eiro et al. Plant Cell. 2003 July; 15(7):1552-62) of Arabidopsis thaliana. EBS has been implicated in the transcriptional repressor complex that modulates chromatin structure and is required to repress the initiation of flowering in short days. Overexpression of EBS caused early flowering in Arabidopsis thaliana plants (Pi{umlaut over (n)}eiro et al. 2003).
5. HON5-Like Polypeptides
High-mobility-group (HMG) proteins are small and relatively abundant chromatin-associated proteins, biochemically defined as small proteins typically around 30 KDa, having a relatively high proportion of basic and acidic amino acids, and capable of solubilising in dilute perchloric or trichloroacetic acid.
Plants and animals possess a family of HMG proteins that are similar on the basis of a shared motif known as the AT-hook, a domain that preferentially recognizes and binds to DNA with certain structural features, such as those imparted by AT-rich DNA. Since these proteins recognize chromatin and/or DNA structure (such as the structure imparted by AT-rich DNA) rather than as specific DNA sequence, they have been named architectural transcription factors.
Much of the information available on the function of the animal HMGA family has been inferred to the plant HMG-1/Y family of AT-hook proteins.
In plants, two groups of chromosomal HMG proteins have been identified, namely the HMGA family, typically containing four A/T-hook DNA-binding motifs, and the HMGB family, containing a single HMG-box DNA-binding domain. Both plant and animal AT hook proteins bind AT-rich tracts of DNA in the minor groove, induce DNA bending, and function in the regulation of gene expression. By orchestrating multiple protein-protein and protein-DNA interactions, the HMGA proteins assist the formation of higher-order transcription factor complexes, regulating gene expression (Klosterman et al; Plant Science 162 (2002) 855—866).